Diglycosidase isolated from microorganisms

ABSTRACT

This invention relates to a novel microorganism-derived enzyme having an activity to cut disaccharide glycosides (particularly, β-primeveroside and/or analogous disaccharide glycosides) in disaccharide unit, a method for producing the enzyme, a gene which encodes the enzyme and use of the enzyme. Various components can be formed by the action of this enzyme upon disaccharide glycosides.

TECHNICAL FIELD

This invention relates to a novel microorganism-derived enzyme having anenzyme activity to cut disaccharide glycosides, particularlyβ-primeveroside and/or its analogs, in disaccharide unit, a method forproducing said enzyme, a gene which encodes said enzyme, a vectorcontaining said gene, a transformant transformed with said vector anduse of said enzyme.

BACKGROUND ART

Alcoholic aromas such as geraniol, linalool, benzyl alcohol, 2-phenylalcohol and C₁₃-norterpenoid alcohol as plant aroma components take animportant role in the aroma formation of, for example, flowers, tea,fruits and wine.

Among these aroma components, monosaccharide glycosides such asβ-D-glucopyranoside have been isolated and identified as aromaprecursors of benzyl alcohol and (Z)-3-hexenol.

Recently, the presence of a disaccharide glycoside β-primeveroside(6-O-β-D-xylopyranosyl-β-D-glucopyranoside) or its analogs has beenconfirmed as precursors of fragrant alcohols such as geraniol andlinalool, which seem to be taking an important role regarding the aromaof flowers. The presence of the disaccharide glycoside β-primeverosideand its analogs has also been revealed as precursors of other alcoholicaroma components described above.

In addition to such aromas, the presence of the disaccharide glycosideβ-primeveroside or its analogs has also been found in certainphysiologically active substances such as pigments and pharmacologicalcomponents. For example, it is known that macrozamin in a cycad, etc. iscut by β-primeverosidase in disaccharide unit to form a toxin.

On the other hand, such an enzyme having a function to cleave precursorsof these aroma components and physiologically active components indisaccharide unit has been confirmed only in a small amount in, forexample, tea leaves (JP-A-8-140675; the term “JP-A” as used herein meansan “unexamined published Japanese patent application”), and almost nostudy on its application has been carried out. Recently, it becameapparent that the aglycon cannot be sufficiently released from thesedisaccahride glycoside and analogues thereof by the action of the knownglucosidase. In consequence, great concern has been directed toward thedevelopment of a method by which such an enzyme can be produced on anindustrial scale at low cost without depending on the prior art supplysources such as tea leaves.

DISCLOSURE OF THE INVENTION

With the aim of solving the aforementioned problems, the inventors ofthe present invention have conducted intensive studies searching for itssupply source in microorganisms. That is, as a result of efforts toscreen a microorganism capable of producing an enzyme having theaforementioned function from a broad range of natural sources, we havefound a microorganism which is suitable for fermentation culturing andhas the ability to produce an enzyme having the action to cut thesaccharide moiety of disaccharide glycosides such as β-primeveroside indisaccharide unit, and have isolated and purified said enzyme anddetermined nucleotide sequence of a gene that encodes said enzyme.Thereafter, we have found the enzyme having the aforementioned action ina large number of microorganisms such as molds, yeast, bacteria andactinomycetes and thereby accomplished this invention.

Accordingly, the invention relates to a novel microorganism-derivedenzyme having an enzyme activity to cut disaccharide glycosides,particularly β-primeveroside and/or its analogs, in disaccharide unit, amethod for producing said enzyme, a gene which encodes said enzyme, avector containing said gene, a transformant transformed with said vectorand use of said enzyme.

The enzyme of the present invention is characterized in that it has anactivity to release saccharides in a disaccharide unit from thedisaccharide glycoside by acting on the disaccharide glycoside which canhardly be utilized as the substrate by the known glycosidase. In thepresent specification, the enzyme having such activity is called“diglycosidase”. The diglycosidase of the present invention has not onlythe activity to act on the disaccharide glycoside to release thesaccharides in a disaccharide unit but also cut the glycoside bonding ofthe monoglycoside. Moreover, it has an activity to cut the glycosidebonding of the modified monoglycoside (e.g., acetylglucoside,malonylglucoside, methylglucoside, phosphoglucoside, andamidoglucoside).

The microorganism-derived enzyme of the present invention which has theactivity to act upon a disaccharide glycoside and thereby releasesaccharides in disaccharide unit from said disaccharide glycoside isdifferent from the plant-derived enzyme in terms of physicochemicalproperties and homology of gene sequences.

Next, the present invention is described in detail.

In this connection, results of the measurement of various enzymeactivities carried out in the present invention are shown by valuesobtained by the following methods unless otherwise noted.

(1) Disaccharide Glycoside Degradation Activity

Measurement of the activity was carried out using an automatic chemicalanalyzer (TBA-30R, manufactured by TOSHIBA CORP.). A 30 μl portion ofeach enzyme sample was mixed with 200 μl of acetate buffer solution (pH5.5) containing 2 mM of p-nitrophenyl (pNP) primeveroside as thedisaccharide glycoside substrate to carry out the reaction at 40° C. andat a cycle time of 22.5 seconds, for 9.75 minutes, and then the reactionsolution was mixed with 250 μl of sodium carbonate to measure absorbanceat 412 nm. Measurement of the sample blank was carried out in the samemanner using 20 mM acetate buffer (pH 5.5) instead of the substratesolution.

One unit of the enzyme activity is defined as the amount of enzyme whichincreases the absorbance by a factor of 1 under these conditions.

The pNP-primeveroside used herein can be synthesized for example byallowing pNP-glucoside (manufactured by Merck) to react withxylo-oligosaccharide (manufactured by Wako Pure Chamical Industries)using an enzyme xylosidase (manufactured by Sigma), thereby effectingtransfer of one xylose residue to pNP-glucoside through β-1,6-bonding.

(2) β-Glucosidase Activity

Measurement of the activity was carried out using an automatic chemicalanalyzer (TBA-30R, manufactured by TOSHIBA CORP.). A 10 μl portion ofeach enzyme sample was mixed with 200 μl of acetate buffer solution (pH5.5) containing 2 mM of p-nitrophenyl (pNP) glucoside as the substrateto carry out the reaction at 40° C. and at a cycle time of 22.5 seconds,and then the reaction solution was mixed with 250 μl of sodium carbonateto measure absorbance at 412 nm. Measurement of the sample blank wascarried out in the same manner using 20 mM acetate buffer (pH 5.5)instead of the substrate solution.

One unit of the enzyme activity is defined as the amount of enzyme whichincreases the absorbance by a factor of 1 under these conditions.

In order to obtain a microorganism capable of producing an enzyme havinga diglycosidase activity, the present inventors have examined a broadrange of natural sources and found that several microbial strainsisolated from the natural world can produce an enzyme having saidactivity. The disaccharide glycosides analogous to β-primeveroside aredisaccharides glycosides having glucose on the aglycon side, such asapiofuranosyl-β-D-glucopyranoside andarabinofuranosyl-β-D-glucopyranoside.

The diglycosidase producing microorganisms of the present invention canbe screened, for example, in the following manner. That is, a soilsample solution is inoculated into a separation liquid medium containingeugenylprimeveroside or the like compound as the sole carbon source tocarry out enrichment culturing, the resulting culture broth is spread ona separation agar plate medium having the same composition, and the thusgrown colonies are selected and isolated. A strain having an activity torelease pNP from bypassing disaccharide (e.g., pNP-primeveroside or thelike) can be selected by culturing the thus isolated strains in anappropriate liquid medium.

A diglycosidase producing microorganism can be screened from the thusselected strains using pNP-primeveroside or the like compound as thesubstrate and release of disaccharide as the index.

Main strains isolated by the present inventors were identified byexamining their mycological properties in the light of the followingreferences (1) to (3).

REFERENCES

-   (1) Raper, K. B. and Fennell, D. I., 1965. “The genus Aspergillus”,    Williams & Wilkins, Baltimore.-   (2) Kozakiewicz, Z., 1989. Aspergillus species on stored products.    Mycological Papers, No. 161, CAB International Mycological    Institute.-   (3) Al-Musallam, A., 1980. “Revision of the black Aspergillus    species”, University of Utrecht.

Mycological properties are described in the following.

Identification of Strain A

(1) Growth Condition

Growth Condition

Czapek Agar Medium

Colony size is 48 to 50 mm in diameter (25° C., 7 days), its surface isvelutinous to powdery, hypha is white, formation of conidia is slightlypoor, dull green to grayish green, backside is light yellowish brown tobrown.

Malt Extract Agar Medium

Colony size is 78 to 80 mm in diameter (25° C., 7 days), its surface isvelutinous powdery, hypha is white, formation of conidia is markedlygood, dull green to grayish green, backside is colorless to yellowishwhite. Colony size at 37° C. (3 days) is 73 to 75 mm in diameter. Goodgrowth even at 45° C.

(2) Morphology

Conidial Heads:

Strong columnar form, 48 to 128 μm in length, 16 to 52 μm in diameter,dull green to grayish green.

Conidiophores:

Forms from substrate mycelium, 125 to 800 μm in length (mostly 500 μm orless), 5 to 10 μm in diameter, straight or slight bending, smoothsurface.

Vesicles:

Diameter from 10 to 25 μm, flask shape, forms phialide in upper ⅔.

Metulae:

Not formed.

Phialides:

5.6˜12×2.4˜3.2 μm

Conidia:

Diameter from 2.6 to 3.6 μm, globose to subglobose, echinulate surface.

Ascospores:

Not formed.

The above results show that the strain A belongs to the Aspergillusfumigatus group, because the conidium forming cells are single columnar(metula is not formed), the conidial head is cylindrical and dull greento grayish green, the conidia are globose and ascospore is not formed.In addition, since the conidial head is strong columnar and does notform nodding appearance, the conidia have echinulate surface and most ofthe conidiophores are 500 μm or less, this strain is Aspergillusfumigatus.

Identification of Strains B, C and D

(1) Growth Condition

TABLE 1 Medium Item Strain B Strain C Strain D Czapek Colony diameter 45to 48 mm 47 to 50 mm 46 to 48 mm agar (25° C., 7 days) medium Colonydiameter 80 mm or 80 mm or 80 mm or (25° C., 14 days) more more moreHyphae layer dense, white dense, white dense, white to yellow Formationof good good good conidia Color of conidia dull grayish dull grayishdull grayish brown to brown to brown to black brown black brown blackbrown Backside color white to white white yellow Malt Colony diameter 46to 51 mm 53 to 55 mm 55 to 59 mm extract (25° C.) agar Hyphae layer thinand thin and thin and medium flat, flat, flat, colorless colorlesscolorless Formation of very good very good very good conidia Color ofconidia black to black to black to black brown black brown black brownBackside color colorless colorless colorless

(2) Morphology (Czapek Agar Medium)

TABLE 2 Item Strain B Strain C Strain D Conidial Shape spherical,spherical, radial, spherical, heads radial, sometimes split radial,sometimes split into cylindrical sometimes split into cylindrical formwhen matured into cylindrical form when form when matured matured Size120 to 560 μm 150 to 500 μm 125 to 350 μm Color dull grayish dullgrayish brown dull grayish brown to black to black brown brown to blackbrown brown Conidio- Origin forms from forms from forms from phoressubstrate substrate mycelium substrate mycelium mycelium Length 350 μmto 3 mm 350 μm to 3 mm 350 μm to 2.5 mm Diameter 9 to 20 μm 10 to 22.5μm 12.5 to 20 μm Surface smooth smooth smooth Vesicles Diameter 20 to 80μm 15 (mostly 35) to 30 to 80 μm 80 μm Shape globose globose globoseMetula entire entire entire formation region Metulae Length 20 to 24 μm12 to 22.4 μm 12.8 to 24 μm Diameter 5.6 to 7.2 μm 4.8 to 6.8 μm 5.6 to8 μm Shape globose to globose to globose to subglobose subglobosesubglobose Surface echinulate echinulate echinulate Ascospore not formednot formed not formed

Based on the above results, all of the strains B, C and D belong to theAspergillus niger group, because the conidia forming cells are doublecolumnar (metulae and phialides are formed) and the conidial heads areglobose and blackish. In addition, since the colony diameter becomes 5cm or more by 14 days on the Czapek agar medium, the conidial surface isechinulate (verrucose), the conidium is globose to subglobose shape of 6μm or less and dull grayish brown to black brown and the conidiophore is6 μm or less, these are strains of Aspergillus niger var. niger.

The present inventors also have selected strains belonging to the genusAspergillus from type cultures at random and-examined their ability toproduce diglycosidase. As a result, productivity of the enzyme was alsofound, for example, in Aspergillus niger IFO 4407, Aspergillus niger IAM2020 and Aspergillus fumigatus IAM 2046, etc. In addition, screening ofvarious other microorganisms was also carried out for their ability toproduce diglycosidase. As a result, the diglycosidase activity was foundin various microorganisms such as those belonging to the genusAspergillus, the genus Penicillium, the genus Rhizopus, the genusRhizomucor, the genus Talaromyces, the genus Mortierella, the genusCryptococcus, the genus Microbacterium, the genus Corynebacterium andthe genus Actinoplanes.

The strains which can be used in the present invention are not limitedto the strains described above, and any strain having diglycosidaseproductivity can be used. In addition, mutants of the strains havingdiglycosidase productivity, or various microorganisms or various cells(e.g., yeast cells, bacterial cells, higher plant cells and animalcells) modified by recombinant DNA techniques to have an ability toproduce diglycosidase, in particular, preferably those modified toproduce diglycosidase in great quantity are also included in theproduction method which can be used in the present invention. Whendiglycosidase productivity is added by introducing a diglycosidase gene,the host microorganism may not have the diglycosidase productivity.

When diglycosidase is produced using the aforementioned variousmicroorganisms, methods and conditions suited for the culturing of saidmicroorganisms can be selected, and such methods and conditions are notparticularly limited. For example, culturing method of theaforementioned various strains may be either liquid culturing or solidculturing, but liquid culturing is preferably used. For example, theliquid culturing can be carried out in the following manner.

Any type of medium can be used, provided that a diglycosidase producingmicroorganism can be grown therein. For example, a medium to be used maycontain carbon sources such as glucose, sucrose, gentiobiose, solublestarch, glycerol, dextrin, molasses and organic acids, nitrogen sourcessuch as ammonium sulfate, ammonium carbonate, ammonium phosphate,ammonium acetate, peptone, yeast extract, corn steep liquor, caseinhydrolysate, wheat bran and meat extract and inorganic salts such aspotassium salts, magnesium salts, sodium salts, phosphates, manganesesalts, iron salts and zinc salts. In addition, various inducers can beadded to the medium in order to produce and accumulate diglycosidase.Examples of the inducers to be used include saccharides, preferablygentose (e.g., Gentose #80, Nihon Shokuhin Kako), gentiobiose andgentio-oligosaccharide (e.g., Gentio-oligosaccharide, Wako PureChemicals). Amount of these inducers to be added is not particularlylimited, with the proviso that the amount is effective in increasing theproductivity of diglycosidase to an intended level, but is addedpreferably in an amount of from 0.01 to 5%.

The medium pH is adjusted to a level of approximately from 3 to 8,preferably from about 5 to 6, and the culturing is carried out underaerobic conditions at a culturing temperature of generally from about 10to 50° C., preferably at about 30° C., for a period of from 1 to 15days, preferably from 4 to 7 days. Regarding the culturing method,shaking culture and aerobic submerged culture by a jar fermentor can beused. However, the aforementioned various culture conditions areoptionally changed depending on the microorganisms or cells to becultured as a matter of course, and such conditions are not particularlylimited with the proviso that the diglycosidase of the present inventioncan be produced.

Regarding the isolation and purification of diglycosidase from the thusobtained culture broth, purified primeverosidase can be obtained in theusual way by a combination of centrifugation, UF concentration, saltingout and various types of chromatography such as of an ion exchangeresin.

The culture of the aforementioned microorganism as it is can be used asthe enzyme composition of the present invention. Of course, the culturemay be purified to an appropriate degree of purification depending onthe intended use of the present invention.

The following further describes a gene which encodes amicroorganism-derived enzyme of the present invention having theactivity to act upon a disaccharide glycoside and thereby releasesaccharides from said disaccharide glycoside in disaccharide unit, arecombinant vector which contains said gene, a transformant into whichsaid vector is introduced and a method for producing said enzyme usingsaid transformant.

As the microorganism-derived enzyme of the present invention having theactivity to act upon a disaccharide glycoside and thereby releasesaccharides from said disaccharide glycoside in disaccharide unit, allof the enzymes which can be obtained by the aforementioned productionmethods are included, in which particularly preferred one is apolypeptide which has the amino acid sequence of SEQ ID NO: 8 shown inthe Sequence Listing, wherein one or more amino acid residues of theamino acid sequence may be modified by at least one of deletion,addition, insertion and substitution, and more preferred one is apolypeptide which has the amino acid sequence of SEQ ID NO: 8 shown inthe Sequence Listing.

Examples of the gene which encodes the enzyme of the present inventioninclude a gene which can be obtained from a microorganism capable ofproducing said enzyme by cloning of said gene and a gene which has acertain degree of homology with said gene. Regarding the homology, agene having a homology of at least 50% or more, preferably a gene havinga homology of 80% or more and more preferably a gene having a homologyof 95% or more can be exemplified. The following polynucleotide (DNA orRNA) is desirable as the gene which encodes the enzyme of the presentinvention.

A polynucleotide which comprises a polynucleotide being selected fromthe following polynucleotides (a) to (g) and encoding a polypeptidehaving the activity to act upon a disaccharide glycoside and therebyrelease saccharides from said disaccharide glycoside in disaccharideunit;

(a) a polynucleotide which encodes a polypeptide having the amino acidsequence of SEQ ID NO: 8 shown in the Sequence Listing,

(b) a polynucleotide which encodes a polypeptide having the amino acidsequence of SEQ ID NO: 8 shown in the Sequence Listing, wherein one ormore amino acid residues of the amino acid sequence are modified by atleast one of deletion, addition, insertion and substitution,

(c) a polynucleotide which has the nucleotide sequence of SEQ ID NO: 7shown in the Sequence Listing,

(d) a polynucleotide which has the nucleotide sequence of SEQ ID NO: 7shown in the Sequence Listing, wherein one or more bases of thenucleotide sequence are modified by at least one of deletion, addition,insertion and substitution,

(e) a gene which hybridizes with any one of the aforementionedpolynucleotides (a) to (d) under a stringent condition,

(f) a polynucleotide which has homology with any one of theaforementioned polynucleotides (a) to (d), and

(g) a polynucleotide which is degenerate with respect to any one of theaforementioned polynucleotides (a) to (f).

The gene which encodes the enzyme of the present invention can beprepared from the aforementioned microorganism capable of producing theenzyme of the present invention by carrying out cloning of said gene inthe following manner. Firstly, the enzyme of the present invention isisolated and purified from a microorganism capable of producing theenzyme of the present invention by the aforementioned method andinformation on its partial amino acid sequence is obtained.

Regarding the determination method of a partial amino acid sequence, itis effective to carry out a method in which purified enzyme is directlyapplied to an amino acid sequence analyzer (such as Protein Sequenser476A, manufactured by Applied Biosystems) by Edman degradation method[J. Biol. Chem., vol. 256, pp. 7990–7997 (1981)], or a method in whichlimited hydrolysis of the enzyme is carried out using a proteinhydrolase, the thus obtained peptide fragments are isolated and purifiedand then amino acid sequences of the thus purified peptide fragments areanalyzed.

Based on the information of the thus obtained partial amino acidsequences, a gene which encodes the enzyme of the present invention iscloned. In general, the cloning is carried out making use of a PCRmethod or a hybridization method.

When a hybridization method is used, the method described in “MolecularCloning, A Laboratory Manual” (edit. by T. Maniatis et al., Cold SpringHarbor Laboratory, 1989) may be used.

When a PCR method is used, the following method may be used.

Firstly, a gene fragment of interest is obtained by carrying out PCRreaction using genomic DNA of a microorganism capable of producing theenzyme of the present invention as the template and syntheticoligonucleotide primers designed based on the information of partialamino acid sequences. The PCR method is carried out in accordance withthe method described in “PCR Technology” (edit. by Erlich H. A.,Stockton Press, 1989). When nucleotide sequences of the thus amplifiedDNA fragments are determined by a usually used method such as thedideoxy chain termination method, a sequence which corresponds to thepartial amino acid sequence of the enzyme of the present invention isfound in the thus determined sequences, in addition to the sequences ofsynthetic oligonucleotide primers, so that a part of the enzyme gene ofinterest of the present invention can be obtained. As a matter ofcourse, a gene which encodes complete enzyme of the present inventioncan be cloned by further carrying out a cloning method such as thehybridization method using the thus obtained gene fragment as a probe.

In the following Examples, a gene coding for the enzyme of the presentinvention was determined by the PCR method using Aspergillus fumigatusIAM 2046. Complete nucleotide sequence of the gene coding for the enzymeof the present invention originated from Aspergillus fumigatus is shownin the SEQ ID NO: 7, and the amino acid sequence encoded thereby wasdetermined to be the sequence shown in the SEQ ID NO: 8. In thisconnection, there are countless nucleotide sequences which correspond tothe amino acid sequence shown in the SEQ ID NO: 8, in addition to thenucleotide sequence shown in the SEQ ID NO: 8, and all of thesesequences are included in the scope of the present invention.

The gene of interest can also be obtained by chemical synthesis based onthe information of the amino acid sequence shown in the SEQ ID NO: 8 andthe nucleotide sequence shown in the SEQ ID NO: 7 (cf. Gene, 60(1),115–127 (1987)).

Regarding the gene of the object enzyme of the present invention, apolynucleotide which encodes a polypeptide having the amino acidsequence of SEQ ID NO: 8, wherein one or more amino acid residues of theamino acid sequence are modified by at least one of deletion, addition,insertion and substitution, a gene which hybridizes with saidpolynucleotide under a stringent condition, a polynucleotide which hashomology with said polynucleotide and a polynucleotide which isdegenerate with respect to said polynucleotide are also included in thepresent invention, with the proviso that the polypeptides encodedthereby have the enzyme activity of the present invention.

The term “under stringent condition” as used herein means, for example,the following condition. That is, 6×SSC, 1.0% blocking agent, 0.1%N-lauroylsarcosine sodium, 0.02% SDS.

By using the entire portion or a part of the enzyme gene of the presentinvention, whose complete nucleotide sequence has been revealed makinguse of Aspergillus fumigatus IAM2046, as a probe for hybridization, DNAfragments having high homology with the enzyme gene of the presentinvention shown in SEQ ID NO: 7 can be selected from genomic DNAlibraries or cDNA libraries of microorganisms capable of producing otherenzymes of the present invention.

The hybridization can be carried out under the aforementioned stringentcondition. For example, DNAs from a genomic DNA library or a cDNAlibrary obtained from a microorganism capable of producing an enzyme ofthe present invention is fixed on nylon membranes, and the thus preparednylon membranes are subjected to blocking at 65° C. in apre-hybridization solution containing 6×SSC, 0.5% SDS, 5× Denhart's and100 μg/ml of salmon sperm DNA. Thereafter, each probe labeled with ³²Por digoxigenin is added thereto, followed by incubation overnight at 68°C. The thus treated nylon membranes are washed in 6×SSC containing 0.1%SDS at room temperature for 10 minutes, in 6×SSC containing 0.1% SDS 45°C. for 30 minutes and then subsequently subjecting the thus washedmembranes to an auto-radiography or detection of digoxigenin to detect aDNA fragment which hybridizes with the probe in a specific fashion.Also, genes which show various degree of homology can be obtained bychanging certain conditions such as washing or lowering thehybridization temperature (e.g., 45° C.).

On the other hand, primers for use in the PCR reaction can be designedfrom the nucleotide sequence of the gene of the present invention. Bycarrying out the PCR reaction using these primers, gene fragments havinghigh homology with the gene of the present invention can be detected andthe complete gene can also be obtained.

In order to determine whether the thus obtained gene encodes apolypeptide having the enzyme activity of interest, the thus determinednucleotide sequence is compared with the nucleotide sequence coding forthe enzyme of the present invention or with its amino acid sequence, andthe identity is estimated based on the gene structure and homology.Alternatively, it is possible to determine whether the gene encodes apolypeptide which has the enzyme activity of interest by producing apolypeptide encoded by the gene and measuring its enzyme activity.

The following method is convenient for producing a polypeptide havingthe enzyme activity of the present invention using the enzyme gene ofthe present invention.

Firstly, transformation of a host is carried out using a vectorcontaining the object gene of the enzyme of the present invention andthen culturing of the thus obtained transformant is carried out undergenerally used conditions, thereby allowing the strain to produce apolypeptide having the enzyme activity of the present invention.

Examples of the host to be used include microorganisms, animal cells andplant cells. Examples of the microorganisms include bacteria such asEscherichia coli and other bacteria belonging to the genera Bacillus,Streptomyces, and Lactococcus, yeasts such as those belonging to thegenera Saccharomyces, Pichia and Kluyveromyces and filamentous fungisuch as those belonging to the genera Aspergillus, Penicillium,Trichoderma and Rhizopus. Examples of the animal cells include thosewhich unitize the baculovirus expression system.

Confirmation of the expression and expressed product can be made easilyby the use of an antibody specific for the enzyme of the presentinvention, and the expression can also be confirmed by measuring theenzyme activity of the present invention.

As described in the foregoing, purification of the enzyme of the presentinvention from the transformant culture medium can be carried out byoptional combination of centrifugation, UF concentration, salting outand various types of chromatography such as of ion exchange resins.

In addition, since the primary structure and gene structure of theenzyme of the present invention have been revealed by the presentinvention, it is possible to obtain a gene coding for the amino acidsequence wherein one or more amino acid residues of the amino acidsequence are modified by at least one of deletion, addition, insertionand substitution, by introducing random mutation or site-specificmutation using the gene of the present invention. This method renderspossible preparation of a gene coding for an enzyme of the presentinvention which has the enzyme activity of the present invention but itsproperties such as optimum temperature, temperature stability, optimumpH, pH stability and substrate specificity are slightly changed, and italso renders possible production of such enzymes of the presentinvention by means of genetic engineering techniques.

Examples of the method for introducing random mutation include achemical DNA modification method in which a transition mutation isinduced to convert cytosine base into uracil base by the action ofsodium hydrogensulfite [Proceedings of the National Academy of Sciencesof the USA, vol. 79, pp. 1408–1412 (1982)], a biochemical method inwhich base substitution is induced during the step of double strandformation in the presence of [α-S] dNTP [Gene, vol. 64, pp. 313–319(1988)] and a PCR method in which PCR is carried out by adding manganeseto the reaction system to decrease fidelity of the nucleotideincorporation [Analytical Biochemistry, vol. 224, pp. 347–353 (1995)].

Examples of the method for introducing site-specific mutation include amethod in which amber mutation is employed [gapped duplex method;Nucleic Acids Research, vol. 12, no. 24, pp. 9441–9456 (1984)], a methodin which recognition sites of restriction enzymes are used [AnalyticalBiochemistry, vol. 200, pp. 81-88 (1992); Gene, vol. 102, pp. 67–70(1991)], a method in which mutation of dut (dUTPase) and ung (uracil DNAglycosylase) is used [Kunkel method; Proceedings of the National Academyof Sciences of the USA, vol. 82, pp. 488–492 (1985)], a method in whichamber mutation is induced using DNA polymerase and DNA ligase[oligonucleotide-directed dual amber (ODA) method: Gene, vol. 152, pp.271–275 (1995); JP-A-7-289262], a method in which a host introduced witha DNA repair system is used (JP-A-8-70874), a method in which a proteincapable of catalyzing DNA chain exchange reaction is used(JP-A-8-140685), a method in which PCR is carried out using twodifferent primers for mutation use to which recognition sites ofrestriction enzymes are added (U.S. Pat. No. 5,512,463), a method inwhich PCR is carried out using a double-stranded DNA vector having aninactivated drug resistance gene and two different primers [Gene, vol.103, pp. 73–77 (1991)] and a method in which PCR is carried out makinguse of amber mutation (WO 98/02535).

Also, site-specific mutation can be introduced easily by the use ofcommercially available kits. Examples of such kits include MUTAN®-GMutagenesis Kit (manufactured by Takara Shuzo) in which the gappedduplex method is used, MUTAN®-K Mutagenesis Kit (manufactured by TakaraShuzo) in which the Kunkel method is used, MUTAN®-Express Km MutagenesisKit (manufactured by Takara Shuzo) in which the ODA(Oligonucleotide-directed Dual Amber) method is used and QUICKCHANGE®Site-Directed Mutagenesis Kit (manufactured by STRATAGENE) in whichprimers for mutation use and Pyrococcus furiosus DNA polymerase areused, as well as TaKaRa BIOMEDICALS LA PCR in vitro Mutagenesis Kit(manufactured by Takara Shuzo) and MUTAN®-Super Express Km in vitroMutagenesis Kit (manufactured by Takara Shuzo) (based on ODA methodutilizing the advantage of LA (Long and Accurate) PCR technology) askits in which PCR is used.

Thus, the primary structure and gene structure of the enzyme of thepresent invention provided by the present invention render possibleproduction of an inexpensive and high purity polypeptide having theenzyme activity of the present invention by means of genetic engineeringtechniques.

In this connection, various literature and references are cited in thespecification, and all of them are incorporated herein by references.

Next, various applications of the enzyme composition of the presentinvention are described.

Diglycosidase can be used for the improvement of various components suchas aromas, colors and physiologically active contents of plant materialsand for adjusting extraction efficiency of these components. Inconsequence, it can be used in the production of food and drinks havingincreased aromas and of spices, perfumes and liquid scents havingincreased aromas, and it also can be used for the early stage release ofunfavorable odor by optionally using it during a step of the justdescribed productions. Regarding the colors, it can be used for theimprovement of hues of plant materials, food and drinks, development ofcolors and production of pigments.

In addition, similar to the case of aromatic components, it can be usedfor the degradation and removal of pigment precursors which are notdesirable in view of qualities, and regarding the physiologicalactivities, it can be used for the increase of pharmacologicalcomponents and useful physiologically active components of crude drugs,herbs and other plant components or degradation and removal ofundesirable components.

That is, it is possible to produce the aforementioned actions byallowing the diglycosidase of the present invention to act upon variousdisaccharide glycoside components.

In addition, the diglycosidase of the present invention may beadministered with the aforementioned physiologically active substance,etc. after mixing or without mixing but by simultaneously or with ashort interval administration, in order for the physiologically activesubstance to be absorbed efficiently into the body, etc.

Examples of the materials containing disaccharide glycosides to betreated by the present invention include those which undergo the actionof diglycosidase, such as foods, cosmetics, medicaments, quasi drugs,agricultural chemicals and feeds, more illustratively, it can also beapplied to the production of industrial products having various aromas,such as foods, toiletries, woodworks and mats produced from plantmaterials.

Food articles having aromatic components can be exemplified as materialsto which the diglycosidase of the present invention is preferablyapplied. As illustrative examples, it can be used in the so-called“wilting” step during the production of oolong tea and jasmine tea andfor the improvement of aromas of black tea (for tea pack by CTC method)and wine. It can also be used for the maintenance of aromas of cosmeticsand liquid scents and improvement of aromas and pharmacological effectsof medicaments.

The diglycosidase of the present invention is also useful in theproduction of pigments. For example, extraction of alizarine dye fromRubia tinctorum L. ruberythric acid can be carried out more efficientlythan the conventional method by the use of the enzyme.

Also, it is possible to produce precursors of disaccharide componentssuch as an aroma, a pigment, a physiologically active component andprimeverose making use of the action of diglycosidase. Improvement ofthe stability and keeping quality of these components, theirdetoxication and modification of pharmacological components for DDS canbe expected by their glycosylation.

In addition, diglycosidase can degrade modified glucosides such asacetylglucoside, malonylglucoside, methylglucoside, phosphoglucoside,and amidoglucoside which can hardly be utilized by glucosidase as itssubstrate more efficiently than known glucosidase. Making use of thisproperty, absorption and yield of isoflavone contained in soybean can beimproved by converting acetylglucoside and malonylglucoside ofisoflavone into their aglycon forms.

The enzyme solution of the present invention may be sprayed to the cutflowers or may be abosorbed by the cut flowers to enhance the aroma offlowers.

Regarding application methods of diglycosidase, its adding method,adding amount, reaction method and the like can be changed at willdepending on the conditions of material to be treated.

Regarding an illustrative application method, the diglycosidase of thepresent invention is added to a plant extract or fermentation productcontaining an aroma precursor, and the mixture is incubated. Thereaction conditions are not particularly limited, with the proviso thatthe diglycosidase of the present invention can act upon the precursor ofan aroma, pigment or physiologically active component to release thearoma, pigment or physiologically active component, and such conditionscan be set by those skilled in the art without undue efforts. Under suchconditions, concentration of said component can be increased.

Also, the enzyme of the present invention can be used for increasingconcentration of an aroma, pigment or physiologically active componentwhich is present in plants. That is, since plants contain precursors ofthese components, an aroma, pigment or physiologically active componentin a plant can be increased by cultivating the plant with adding aneffective amount of the diglycosidase of the present invention(including transgenic method) under such conditions that the precursorin said plant can be hydrolyzed. In addition, the formation period of anaroma, pigment or physiologically active component can be controlledmaking use of the enzyme composition of the present invention.

In this connection, it is possible to synthesize various types ofglycoside by making use of reverse reaction of the diglycosidase of theinvention.

BEST MODE FOR CARRYING OUT THE INVENTION

The invention is described further in detail in the following withreference to examples but, as a matter of course, the invention is notlimited to the following examples without departing from its scope.Unless otherwise noted, the term % as used herein means w/v %.

EXAMPLE 1

Each of Aspergillus niger IFO 4407 and Aspergillus niger IAM 2020 wascultured overnight at 30° C. on a shaker in a pre-culture medium(composition; 0.2% yeast extract, 0.5% peptone, 2% glucose, 0.1% KH₂PO₄,0.05% MgSO₄.7H₂O, pH 5.7), the resulting culture broth in an amount of1/100 was inoculated into a main culture medium (4% soybean flour, 0.3%sodium chloride, 0.1% KH₂PO₄, 0.05% MgSO₄.7H₂O, 2% soluble starch, 1%red bran, pH 5.6), and cultured for 6 days on a shaker, and then thecells were removed from the culture broth to obtain a crude enzymesolution. Using this enzyme solution, diglycosidase activity andβ-glucosidase activity were measured.

As the result, the diglycosidase activity and β-glucosidase activity inthe strain IFO 4407 were 0.129 unit/ml and 4.34 units/ml, respectively,and the diglycosidase activity and β-glucosidase activity in the strainIAM 2020 were 0.156 unit/ml and 5.97 units/ml, respectively.

EXAMPLE 2

In accordance with Example 1, Aspergillus fumigatus IAM 2046 waspre-cultured in the same manner, and the resulting culture broth wasinoculated into a main culture medium (2% soybean flour, 0.3% sodiumchloride, 0.1% KH₂PO₄, 0.05% MgSO₄.7H₂O, 3% soluble starch, 0.5% gentose#80 (mfd. by Nihon Shokuhin Kako), pH 5.6) and cultured for 4 days toobtain a crude enzyme solution. As the result, the diglycosidaseactivity was 0.106 unit/ml and β-glucosidase activity was 0.320 unit/ml.

EXAMPLE 3

Using Aspergillus fumigatus IAM 2046, effects of inducers on theproduction of diglycosidase were examined. Aspergillus fumigatus IAM2046 was cultured for 6 days in a culture medium (2% soybean flour, 0.3%sodium chloride, 0.1% KH₂PO₄, 0.05% MgSO₄.7H₂O, 3% soluble starch)supplemented with 0.1% of each of various saccharides, and thediglycosidase activity was measured. The results are shown in Table 3.

TABLE 3 Inducers Inducing ability (%) Not added 100 Isomaltose 145Maltotriose 171 Maltose 136 Gentose #80 235 Gentiobiose 211Gentio-oligosaccharide 180 Sucrose 116 Trehalose 113 Glucose 164Galactose 125 Fructose 143 Rhamnose 129 Tulbose 116 Maltitol 142Arabitol 112 Galactitol 142 Glucosamine hydrochloride 157

As is evident from the above table, the producing ability ofdiglycosidase was increased by various saccharides. Particularly,markedly high inducing ability was found in gentose, gentiobiose andgentio-oligosaccharide.

In addition, similar effects were also found when Aspergillus niger IFO4407 or Aspergillus niger IAM 2020 was used.

EXAMPLE 4

Each of the crude enzyme solutions obtained in Examples 1 and 2 wasconcentrated using an ultrafiltration membrane having a molecular weightcutoff of 6,000. Next, 1 ml of the concentrated solution was mixed with1 ml of a 5 mg/ml pNP-primeveroside solution which had been preparedusing 20 mM phosphate buffer (pH 6.0), and the mixture was incubated at37° C. Samples were collected 1, 2, 4, 24 and 48 hours thereafter toconfirm release of primeverose by a thin layer chromatography (TLC).

As a result, a spot was observed by TLC at the same position of adisaccharide primeverose in all of the culture media of two Aspergillusniger strains described in Example 1 and Aspergillus fumigatus describedin Example 2. Such a spot was not observed in samples in which the crudeenzyme concentration solutions were subjected to the same test aftertheir heat treatment (100° C. for 10 minutes). Thus, the presence of anenzyme capable of releasing primeverose in disaccharide unit frompNP-primeveroside was found in the crude enzyme concentration solutions.

EXAMPLE 5

Screening of Diglycosidase in Various Microorganisms

a) Preparation of Enzyme Samples

Each microorganism to be subjected to screening was pre-cultured andmain cultured. In the case of liquid culturing, the obtained culturebroth was centrifuged at 10,000 min⁻¹ for 10 minutes and the resultingsupernatant was used as the enzyme sample. In the case of solidculturing, the medium after completion of the culturing was extractedwith water and the resulting extract was used as the enzyme sample.

In this connection, intracellular enzyme was also examined in the caseof bacteria. In that case, the culture broth was centrifuged, and thethus obtained cells as the precipitate were washed with physiologicalsaline, suspended in 10 times amount of 10 mM phosphate buffer (pH 7.0)based on the cell weight and then treated with ultrasonic wave todisrupt the cells. The disrupted suspension was centrifuged at 12,000min⁻¹ for 20 minutes and the resulting supernatant was used as theintracellular enzyme sample.

Media and culture conditions regarding the culturing are shown in Tables1 to 5.

TABLE 1 Liquid culturing of mold and yeast Pre-culture: Mediumcomposition Yeast extract (DIFCO) 0.2% Peptone (DIFCO) 0.5% Glucose(Katayama Chemical) 2.0% Potassium dihydrogenphosphate 0.1% (KantoChemical) Magnesium sulfate heptahydrate 0.05% (Katayama Chemical)

The above composition was dissolved in purified water and adjusted to pH5.7 with 1 M hydrochloric acid and 1 M sodium hydroxide. The medium wasdispensed in 100 ml portions into Sakaguchi flasks and then sterilizedat 121° C. for 20 minutes under 1 atmospheric pressure.

Pre-Culture: Culture Conditions

The culturing was carried out at a shaking speed of 140 min⁻¹, with oneloopful inoculum from a slant culture, for 1 day or more and at 30° C.Regarding yeast strains, temperature condition was set to 25° C.

Main culture: Medium composition Soya flower A (Nisshin) 2.0% Sodiumchloride (Kanto Chemical) 0.3% Dipotassium hydrogenphosphate 0.1% (KantoChemical) Magnesium sulfate heptahydrate 0.05% (Katayama Chemical)Soluble starch (Wako Pure Chemical) 3.0% Gentose #80 (Nihon ShokuhinKako) 0.5%

The above composition was dissolved in purified water and adjusted to pH5.6 with 1 M hydrochloric acid and 1 M sodium hydroxide. The medium wasdispensed in 100 ml portions into Sakaguchi flasks and then sterilizedat 121° C. for 20 minutes under 1 atmospheric pressure.

The culturing was carried out using the medium both in the presence andabsence of gentose #80.

Main Culture: Culture Conditions

The culturing was carried out at a shaking speed of 140 min⁻¹, with 1 mlinoculum from the pre-culture broth, for 5 days and at 30° C. Regardingyeast strains, the temperature condition was set to 25° C.

TABLE 2 Solid culturing of mold and yeast Pre-culture: Mediumcomposition High starch bran 8.3% (B Ohgi, Nippon Flower Milling)

The above composition was suspended in purified water, and the mediumwas dispensed in 9 ml portions into culture test tubes and thensterilized at 121° C. for 20 minutes under one atmospheric pressure.

Pre-Culture: Culture Conditions

The culturing was carried out at a shaking speed of 300 min⁻¹, with oneloopful inoculum from a slant culture, for 1 to 2 days and at 30° C.

Main Culture: Medium Composition

A 5.0 g portion of bran was suspended in 1.5 ml of purified water,dispensed into 100 ml capacity conical flasks and then sterilized at121° C. for 20 minutes under one atmospheric pressure.

Main Culture: Culture Conditions

The culturing was carried out with 1 ml inoculum from the pre-culturebroth, for 3 days and at 30° C.

Extraction

By adding 90 ml of tap water, extracted overnight at 8° C. or below.

TABLE 3 Culturing of bacteria and actinomycetes Pre-culture: Mediumcomposition Tryptic soy broth (DIFCO) BACTO Tryptone 1.7% BACTO Soytone0.3% BACTO Dextrose 0.2% Sodium chloride 0.5% {close oversize brace} pH7.3 ± 0.2 Dipotassium 0.25% hydrogenphosphate

The above composition was dissolved in purified water, and the mediumwas dispensed in 100 ml portions into Sakaguchi flasks and thensterilized at 121° C. for 20 minutes under one atmospheric pressure.

Pre-Culture: Culture Conditions

The culturing was carried out at a shaking speed of 140 min⁻¹, with oneloopful inoculum from a slant culture, for 1 day or more and at 30° C.

Main culture: Medium composition Polypeptone (Japan Pharmaceutical) 1.0%Yeast extract (DIFCO) 0.25% Ammonium sulfate (Wako Pure Chemical) 0.1%Dipotassium hydrogenphosphate 0.05% (Kanto Chemical) Magnesium sulfateheptahydrate 0.025% (Katayama Chemical) Calcium chloride (Wako PureChemical) 0.0001% Adekanol LG126 (Asahi Denka) 0.001% Gentose #80 (NihonShokuhin Kako) 0.5%

The above composition was dissolved in purified water and adjusted to pH7.0 with 1 M hydrochloric acid and 1 M sodium hydroxide. The medium wasdispensed in 100 ml portions into Sakaguchi flasks and then sterilizedat 121° C. for 20 minutes under one atmospheric pressure.

The culturing was carried out using the medium both in the presence andabsence of gentose #80.

Main Culture: Culture Conditions

The culturing was carried out at a shaking speed of 140 min⁻¹, with 1 mlinoculum from the pre-culture broth, for 5 days and at 30° C.

TABLE 4 Culturing of Penicillium multicolor Pre-culture: Mediumcomposition Defatted soybean “Soypro” (Hohnen Oil) 2.0% Glucose(Katayama Chemical) 3.0% Potassium dihydrogenphosphate 0.5% (KantoChemical) Ammonium sulfate (Wako Pure Chemical) 0.4% Dry yeast 0.3%Adekanol (Asahi Denka) 0.05%

The above composition was dissolved in purified water, and the mediumwas dispensed in 100 ml portions into Sakaguchi flasks and thensterilized at 121° C. for 20 minutes under one atmospheric pressure.

Pre-Culture: Culture Conditions

The culturing was carried out at a shaking speed of 140 min⁻¹, with oneloopful inoculum from a slant culture, for 5 days or more and at 27° C.

Main culture: Medium composition Gentose #80 (Nihon Shokuhin Kako) 3.0%Potassium dihydrogenphosphate 2.0% (Kanto Chemical) Ammonium sulfate(Wako Pure Chemical) 1.0% Meast P1G (Asahi Beer Food) 3.13% AdekanolLG126 (Asahi Denka) 0.05%

The above composition was dissolved in purified water, and the mediumwas dispensed in 100 ml portions into Sakaguchi flasks and thensterilized at 121° C. for 20 minutes under one atmospheric pressure.

Main Culture: Culture Conditions

The culturing was carried out at a shaking speed of 140 min⁻¹, with 1 mlinoculum from the pre-culture broth, for 6 days and at 27° C.

TABLE 5 Culturing of the genus Corynebacterium Pre-culture: Mediumcomposition Glucose 0.2% Yeast extract 0.1% Ammonium nitrate 0.4%Potassium dihydrogenphosphate 0.15% Sodium hydrogenphosphatedodecahydrate 0.15% Magnesium sulfate heptahydrate 0.02% Ferrous sulfateheptahydrate 0.0001% Calcium chloride dihydrate 0.001%

The above composition was dissolved in purified water and adjusted to pH7.0 with 1 M hydrochloric acid and 1 M sodium hydroxide. The medium wasdispensed in 10 ml portions into culture test tubes and then sterilizedat 121° C. for 20 minutes under one atmospheric pressure.

Pre-Culture: Culture Conditions

The culturing was carried out at a shaking speed of 300 min⁻¹, with oneloopful inoculum from a slant culture, for 2 days and at 30° C.

Main culture: Medium composition Eugenyl-β-primeveroside 0.2% Yeastextract 0.1% Ammonium nitrate 0.4% Potassium dihydrogenphosphate 0.15%Sodium hydrogenphosphate dodecahydrate 0.15% Magnesium sulfateheptahydrate 0.02% Ferrous sulfate heptahydrate 0.0001% Calcium chloridedihydrate 0.001%

The above composition was dissolved in purified water and adjusted to pH7.0 with 1 M hydrochloric acid and 1 M sodium hydroxide. The medium wasdispensed in 10 ml portions into culture test tubes and then sterilizedat 121° C. for 20 minutes under one atmospheric pressure.

Main Culture: Culture Conditions

The culturing was carried out at a shaking speed of 140 min⁻¹, with 1 mlinoculum from the pre-culture broth, for 1 day and at 30° C.

b) Preparation of Substrate Solution

pNP-β-primeveroside was dissolved in 20 mM acetate buffer (pH 5.5) to aconcentration of 5 mg/ml and used as a substrate solution A.Eugenyl-β-primeveroside was dissolved in 20 mM acetate buffer (pH 5.5)to a concentration of 10 mg/ml and used as a substrate solution B.

c) Enzyme Reaction

A 100 μl portion of the substrate solution A was put into amicro-centrifugation tube and mixed with 100 μl of each enzyme sample tocarry out 96 hours of the enzyme reaction in a water bath of 37° C. Whenthe reaction reached the intended time, the reaction solution wastreated at 100° C. for 10 minutes to stop the enzyme reaction. This wasused as the enzyme reaction-completed solution.

As a comparative control of the enzyme sample, the same enzyme samplewas treated at 100° C. for 10 minutes before the enzyme reaction andsubjected to the same reaction. The enzyme reaction was carried out oneach of the substrate solutions A and B. When the substrate solution Bwas used, the reaction was carried out in the same manner as the case ofthe substrate solution A.

d) Thin Layer Chromatography

20 μl of the enzyme reaction-completed solution was spotted on a thinlayer of silica gel (Silica gel 60 F254 [1.05554], Merck) and dried.This was developed twice with a developing solvent prepared by mixingethyl acetate, acetic acid and purified water at a ratio of 3:1:1. Aftercompletion of the development, the thin layer was air-dried. Thereafter,a color developing reagent prepared by mixing sulfuric acid and methanolat a ratio of 20:80 was sprayed all over the thin layer after completionof the development, and the color was developed at 105° C. for about 10minutes.

An enzyme sample by which the spot of primeverose was appeared on thethin layer after the color development was judged that theprimeverosidase was present therein, and this producer strain was judgedas a diglycosidase producing strain.

e) Results of the Screening of Diglycosidase Producing Strains

A summary of the diglycosidase producing strains found by the aboveevaluation method is shown in Table 6.

TABLE 6 Strains in which the diglycosidase production was foundMicroorganisms Strain names Mold Aspergillus oryzae IAM 2769 Aspergillusniger IAM 2020 IFO 4091 IFO 9455 IAM 2107 Aspergillus aculeatusPenicillium rugolosum IFO 7242 Penicillium lilacinum IFO 5350Penicillium decumbence IFO 31297 Penicillium multicolor IAM 7153Rhizopus oryzae JCM 5560 Rhizomucor pusillus IAM 6122 Rhizomucor mieheiIFO 9740 Talaromyces emersonii IFO 9747 Mortierella vinacea IFO 7875Yeast Cryptococcus albidus IAM 12205 Bacteria Microbacterium arborescensJCM 5884 Corynebacterium ammoniagenes IFO 12072 Corynebacteriumammoniagenes IFO 12612 Corynebacterium glutamicum IFO 1318 ActinomycetesActinoplanes missouriensis JCM 3121

EXAMPLE 6

Purification of Diglycosidase Derived from Aspergillus fumigatus

As the pre-culture, Aspergillus fumigatus IAM 2046 was inoculated into aglucose-peptone medium (0.2% yeast extract, 0.5% peptone, 2% glucose,0.1% potassium dihydrogenphosphate, 0.05% magnesium sulfate, pH 5.7) andcultured at 30° C. for 24 hours on a shaker. The pre-culture broth wasinoculated in an inoculum size of 1% into the main culture medium (2%Soya flower, 0.3% sodium chloride, 0.1% dipotassium hydrogenphosphate,0.05% magnesium sulfate, 3% soluble starch, 1% gentose #80, pH 5.6) andcultured at 30° C. for 6 days on a shaker.

Cells were removed from the culture broth by filter paper filtration,and 8,600 ml of the resulting filtrate was concentrated to 710 ml usingan ultrafiltration membrane of 6,000 molecular weight cutoff (AIP-1010,mfd. by Asahi Chemical Industry). A 200 ml portion of the concentratedsolution was centrifuged at 4° C. and at 15,000 rpm for 10 minutes, and192 ml of the supernatant was mixed with 55.9 g of ammonium sulfate (50%saturation) and stirred overnight at 4° C. This was centrifuged at 4° C.and at 15,000 rpm for 10 minutes, and the thus obtained precipitate wasdissolved in 10 ml of 20% saturation ammonium sulfate/20 mM phosphatebuffer (pH 6.0) and centrifuged at 4° C. and at 15,000 rpm for 10minutes to recover the supernatant. A 9.5 ml portion of the supernatantwas applied to a Phenyl Sepharose column (16×100 mm, mfd. by Pharmacia)which had been equilibrated with 20% saturation ammonium sulfate/20 mMphosphate buffer (pH 6.0), and the adsorbed protein was released by anammonium sulfate linear density gradient of from 20% to 0%. The activepeaks were recovered, buffer-exchanged to 25 mM triethanolamine buffer(pH 8.3) using 10DG column (mfd. by BIO-RAD), applied to an anionexchange Mono-P column (5×200 mm, mfd. by Pharmacia) which had beenequilibrated with 25 mM triethanolamine buffer (pH 8.3) and then elutedwith Polybuffer (pH 5.0, mfd. by Pharmacia), and the thus adsorbedprotein was released by a pH linear density gradient of from pH 8.3 topH 5.0 to obtain a purified enzyme preparation of diglycosidase. AnSDS-PAGE analysis confirmed that the enzyme was purified as a singleband of 47 kDa. Also, when it was treated with Endoglycosidase H (mfd.by BOEHRINGER MANNHEIM), changes in the band size were not found.

EXAMPLE 7

Physicochemical Properties of Diglycosidase Derived from Aspergillusfumigatus

Its optimum pH was measured in the following manner. A 400 μl portion of2 mM pNP-primeveroside solution which had been adjusted to a respectivepH value of from pH 2 to 5 with 20 mM secondary citric acid-HCl bufferwas incubated at 37° C. for 5 minutes. Next, this was mixed with 90 μlof the enzyme solution to carry out the reaction at 37° C. for 20minutes. The reaction was stopped by adding 500 μl of 0.5 M sodiumcarbonate solution, and the activity measurement was carried out bymeasuring the absorbance at 420 nm. As a result, it was found that itsoptimum pH was from 2.5 to 3.0. It was found that it shows sufficientactivity at pH 3 which is more lower pH value than those ofplant-derived enzymes having similar activity.

Its optimum temperature was measured in the following manner. A 400 μlportion of 2 mM pNP-primeveroside solution prepared using 20 mM disodiumcitric acid-HCl buffer (pH 2.5) was mixed with 90 μl of the enzymesolution to carry out the reaction at 30 to 65° C. for 20 minutes. Thereaction was stopped by adding 500 μl of 0.5 M sodium carbonatesolution, and the activity was determined by measuring the absorbance at420 nm. It was found that sufficient activity is maintained, becausediglycosidase derived from Aspergillus fumigatus has 80% of the activityeven at 60° C., in comparison with the plant-derived enzymes havingsimilar activity.

Its pH stability was measured in the following manner. The purifiedenzyme preparation was diluted 100 times with each of disodium citratebuffer of from pH 2 to 5, phosphate buffer of from pH 6 to 8 or glycineNaCl—NaOH buffer of from pH 7 to 10 and treated at 37° C. for 1 hour,and then a 90 μl portion thereof was mixed with 400 μl of 2 mMpNP-primeveroside solution (pH 2.5) which had been incubated at 37° C.for 5 minutes, and the reaction was carried out at 37° C. for 20minutes. The reaction was stopped by adding 500 μl of 0.5 M sodiumcarbonate solution, and the activity measurement was carried out bymeasuring the absorbance at 420 nm to calculate the residual activity.As a result, its pH stability was 100% at pH 8 and it was stable withina range of from pH 3 to 8. It was found that this enzyme is stablewithin broader pH range in comparison with plant-derived enzymes whichhave similar activity and are stable at pH 4 to 7.

Thermal stability of the purified preparation was examined by dilutingit 100 times with 20 mM glycine NaCl—NaOH buffer (pH 8), treating thedilution at each temperature of from 30 to 55° C. for 1 hour and thenmeasuring the residual activity. As a result, the activity was stable ata temperature of 50° C. or below. It was found that this enzyme wasstable broader range of temperature, in comparison with plant-derivedenzymes which have similar activity and are stable at 45° C. or below.

Physicochemical Properties of Diglycosidase Derived from OtherMicroorganisms

Diglycosidase samples, the production of which had been confirmed by thesame method as described in Example 5, were examined for physicochemicalproperties. As a result, it was found that diglycosidase has an activityenough for practical use at pH 3 or less because of its optimum pHranging from 3 to 6, that it has an activity enough for practical use at50° C. or more because of its optimum temperature ranging from 30° C. to60° C., and that it is stable at pH 3 to 8 and at 50° C. or less. Thus,diglycosidase derived from microorganisms can be used in relativelybroad ranges of pH and temperature in comparison with similar enzymesderived from plants and is superior thereto in stability.

EXAMPLE 8

Isolation of Gene Coding for the Diglycosidase Derived from Aspergillusfumigatus

Unless otherwise noted, gene manipulation techniques employed hereinwere carried out in accordance with a textbook (e.g., Molecular Cloning2nd ed., Cold Spring Harbor Laboratory Press, 1989).

a) Isolation of Chromosomal DNA

Aspergillus fumigatus IAM 2046 was inoculated into a glucose-peptonemedium (0.2% yeast extract, 0.5% peptone, 2% glucose, 0.1% potassiumdihydrogenphosphate, 0.05% magnesium sulfate, pH 5.7) and cultured at30° C. for 3 days on a shaker.

In accordance with the method of Michael J. Hynes (Molecular andCellular Biology, 1983, Vol. 3, No. 8, 1430–1439), 0.2 ml of chromosomalDNA having a concentration of 12.6 mg/ml was obtained from 300 ml of theculture broth.

b) Determination of Partial Amino Acid Sequence

The purified enzyme preparation of diglycosidase obtained in the Examplewas applied to a protein sequencer (mfd. by Hewlett Packard) todetermine the 22 residue N-terminal amino acid sequence shown in SEQ IDNO: 1. Next, the purified enzyme preparation of diglycosidase obtainedin the Example was subjected to reductive carboxylmethylation and thendigested with lysyl endopeptidase. The thus obtained digest was appliedto a reverse phase liquid chromatography, and one of the digestedpeptide fractions was applied to the protein sequencer to determine the22 residue internal amino acid sequence shown in SEQ ID NO: 2.

SEQ ID NO: 1

Ala-Ala-Ser-Ala-Ser-Ala-Tyr-Cys-Ser-Asn-Ser-Ala-Gly-Asn-Tyr-Lys-Leu-Ser-Ser-Ile-Ala-Ala

SEQ ID NO: 2

Leu-Met-Thr-Pro-Ala-Gly-Ala-Asn-Phe-Ala-Leu-Met-Arg-His-Thr-Ile-Gly-Ala-Ser-Asp-Leu-Ser

c) Preparation of DNA Probe by PCR

Based on the N-terminal amino acid sequence and internal amino acidsequence, the following four mixed oligonucleotides were synthesized bya DNA synthesizer and used as PCR primers.

SEQ ID NO: 3

Sense primer:

5′-ACGAATTCAA(TC)(TA)(CG)IGC(TCAG)GGIAA(TC)TA(TC)AA-3′

SEQ ID NO: 4

Sense primer:

5′-CGGAATTCTA(TC)TG(TC)(TA)(CG)IAA(TC)(TA)(CG)IGC(TCAG)GG-3′

SEQ ID NO: 5

Antisense primer:

5′-TCAAGCTTGC(AG)AA(AG)TTIGC(TCAG)CCIGC(TCAG)GG-3′

SEQ ID NO: 6

Antisense primer:

5′-AGAAGCTTGCICC(TAG)ATIGT(AG)TG(TCAG)C(TG)CAT

Using these primers and the Aspergillus fumigatus chromosomal DNA as thetemplate, PCR reaction was carried out under the following conditionsusing GeneAmp PCR System 9600 (Perkin Elmer).

<PCR reaction solution> 10 x PCR reaction buffer (Perkin Elmer) 10 μldNTP mixed solution (each 2 mM, Perkin Elmer) 10 μl 25 mM MgCl₂ (PerkinElmer) 6 μl chromosomal DNA solution (100 μg/ml) 1 μl 40 μM sense primer2.5 μl 40 μM antisense primer 2.5 μl sterilized water 67.5 μl AmplitaqGold (5 U/μl, Perkin Elmer) 0.5 μl

<PCR reaction conditions> Stage 1: denaturation (95° C., 9 minutes) 1cycle Stage 2: denaturation (94° C., 45 seconds) 30 cycles annealing(55° C., 1 minute) elongation (72° C., 2 minutes) Stage 3: elongation(72° C., 10 minutes) 1 cycle

When the thus obtained DNA fragment of about 0.27 kbp was cloned intopUC19 (TOYOBO) and then its nucleotide sequence was examined, anucleotide sequence coding for the partial amino acid sequence describedin the foregoing was found between just after the sense primer and justbefore the antisense primer. This DNA fragment was used as the DNA probefor the gene cloning.

d) Preparation of Gene Library

By recovering total RNA from Aspergillus fumigatus, Poly(A) RNA wasprepared using Poly(A)Quick mRNA Isolation Kit (mfd. by STRATAGENE).Next, cDNA was synthesized using ZAP-cDNA Synthesis Kit (mfd. bySTRATAGENE), ligated to λZAP II vector (mfd. by STRATAGENE) and thensubjected to packaging using Gigapack III Gold (mfd. by STRATAGENE) toobtain a gene library.

e) Screening of Gene Library

The 0.27 kbp DNA fragment obtained in the aforementioned step c) waslabeled using DIG-High Prime (mfd. by BOEHRINGER MANNHEIM). Using thisas the DNA probe, the gene library obtained in the step d) was screenedby plaque hybridization. After recovering phage particles from the thusobtained positive plaques, a plasmid pAFPri containing a cDNA of about1.7 kbp was obtained by the in vivo excision method in accordance withthe instruction of STRATAGENE.

f) Determination of Nucleotide Sequence

A nucleotide sequence coding for the diglycosidase is shown in SEQ IDNO: 7. Also, an amino acid sequence encoded by the SEQ ID NO: 7 is shownin SEQ ID NO: 8. Since the N-terminal amino acid sequence (SEQ ID NO: 1)and internal amino acid sequence (SEQ ID NO: 2) determined in the stepb) were found in this amino acid sequence, it was confirmed that thisDNA fragment is a diglycosidase gene fragment.

SEQ ID NO: 7 gccgcctctg cttcggctta ctgttccaac tcggccggca actacaagctgtcctccatc 60 gcagctccgg ttcaaggggc cggaaacccc ggctcggaat cgacctggcaattgaccgtt 120 gacgacactt cgtccggtca caaacagacg atagttgggt tcggtgctgctgtcactgat 180 gccacggtca cctcgttcaa cactttgtcc gcctccgtgc tgcaagacttgctcaataaa 240 ctgatgacac ctgccggggc gaactttgct ttgatgcgac atactattggggcttcggat 300 ctgtccggtg acccagccta cacgtacgat gacaatggtg ggaaagcggatccgtcactg 360 tcgggattca acctggggga ccgcggaacg gctatggcca agatgttggcaacaatgaag 420 tctctgcagc ccaacctcaa gatcctcggc tctccctgga gtgcaccaggatggatgaag 480 ctgaacgggg tccttgatgg caatacgaac aacaacaact tgaacgatggatacctaacc 540 agtgggggaa ccggtagtac ggggtatgcc agtcaattcg cgcagtactttgtcaagtac 600 attcaggcct ataagaatct cggtgctcac gtcgacgcga ttaccatccagaacgagccg 660 ctgttcagct cagcgggcta tcccaccatg tatgtctacg attatgagtcggcacagctg 720 atccagaact acatcggccc cgctcttgcc agcgcggggc tagatacggaaatctgggct 780 tatgaccaca acacagatgt cccgtcgtac ccccagactg tccttaaccaggccggtcag 840 tacgtcaagt cggtggcctg gcactgctac gctcccaacg tcgactggaccgtgctcagc 900 cagttccaca acacaaaccc tggagtgaag caatatatga ccgagtgctggactccagca 960 tctggcgcat ggcatcaggc ggcggacttc accatgggtc ccctgcagaactgggcctcg 1020 ggagtggcag catggactct gggaaccaac gctcaggatg gtccgcatctgtccactggc 1080 ggctgcgcga catgtcaagg cttggtgacc atcaacaacg gaggatacacgctcaacacc 1140 gcatactaca tgatggcgca attcagcaag ttcatgccgc ctggtgcgattgtgctcaat 1200 ggcagtggca gctacacgta ctctggcgga ggcggtatcc agtccgtggcttccttgaat 1260 cccgatggaa cccgcactgt ggttattgaa aacacttttg gcaatgatgtctatgtgact 1320 gtcactatga agagcgggca gaagtggagt gggaacgccc ctagccaatccgtgactacc 1380 tgggttcttc catctgcttg a 1401 SEQ ID NO: 8 Ala Ala SerAla Ser Ala Tyr Cys Ser Asn Ser Ala Gly Asn Tyr Lys   1               5                 10                  15 Leu Ser Ser Ile Ala Ala Pro ValGln Gly Ala Gly Asn Pro Gly Ser              20                  25                 30 Glu Ser Thr Trp Gln Leu Thr Val Asp Asp Thr Ser SerGly His Lys          35                  40                  45 Gln ThrIle Val Gly Phe Gly Ala Ala Val Thr Asp Ala Thr Val Thr      50                 55                  60 Ser Phe Asn Thr Leu Ser Ala SerVal Leu Gln Asp Leu Leu Asn Lys  65                  70                 75                  80 Leu Met Thr Pro Ala Gly Ala AsnPhe Ala Leu Met Arg His Thr Ile                  85                  90                 95 Gly Ala Ser Asp Leu Ser Gly Asp Pro Ala Tyr Thr TyrAsp Asp Asn             100                 105                 110 GlyGly Lys Ala Asp Pro Ser Leu Ser Gly Phe Asn Leu Gly Asp Arg         115                120                 125 Gly Thr Ala Met Ala Lys Met LeuAla Thr Met Lys Ser Leu Gln Pro     130                 135                140 Asn Leu Lys Ile Leu Gly Ser Pro Trp Ser Ala Pro GlyTrp Met Lys 145                 150                 155                160 Leu Asn Gly Val Leu Asp Gly Asn Thr Asn Asn Asn AsnLeu Asn Asp                 165                 170                 175Gly Tyr Leu Thr Ser Gly Gly Thr Gly Ser Thr Gly Tyr Ala Ser Gln            180                 185                 190 Phe Ala Gln TyrPhe Val Lys Tyr Ile Gln Ala Tyr Lys Asn Leu Gly         195                200                 205 Ala His Val Asp Ala Ile Thr IleGln Asn Glu Pro Leu Phe Ser Ser     210                 215                220 Ala Gly Tyr Pro Thr Met Tyr Val Tyr Asp Tyr Glu SerAla Gln Leu 225                 230                 235                240 Ile Gln Asn Tyr Ile Gly Pro Ala Leu Ala Ser Ala GlyLeu Asp Thr                 245                 250                 255Glu Ile Trp Ala Tyr Asp His Asn Thr Asp Val Pro Ser Tyr Pro Gln            260                 265                 270 Thr Val Leu AsnGln Ala Gly Gln Tyr Val Lys Ser Val Ala Trp His         275                280                 285 Cys Tyr Ala Pro Asn Val Asp TrpThr Val Leu Ser Gln Phe His Asn     290                 295                300 Thr Asn Pro Gly Val Lys Gln Tyr Met Thr Glu Cys TrpThr Pro Ala 305                 310                 315                320 Ser Gly Ala Trp His Gln Ala Ala Asp Phe Thr Met GlyPro Leu Gln                 325                 330                 335Asn Trp Ala Ser Gly Val Ala Ala Trp Thr Leu Gly Thr Asn Ala Gln            340                 345                 350 Asp Gly Pro HisLeu Ser Thr Gly Gly Cys Ala Thr Cys Gln Gly Leu         355                360                 365 Val Thr Ile Asn Asn Gly Gly TyrThr Leu Asn Thr Ala Tyr Tyr Met     370                 375                380 Met Ala Gln Phe Ser Lys Phe Met Pro Pro Gly Ala IleVal Leu Asn 385                 390                 395                400 Gly Ser Gly Ser Tyr Thr Tyr Ser Gly Gly Gly Gly IleGln Ser Val                 405                 410                 415Ala Ser Leu Asn Pro Asp Gly Thr Arg Thr Val Val Ile Glu Asn Thr            420                 425                 430 Phe Gly Asn AspVal Tyr Val Thr Val Thr Met Lys Ser Gly Gln Lys         435                440                 445 Trp Ser Gly Asn Ala Pro Ser GlnSer Val Thr Thr Trp Val Leu Pro     450                 455                460 Ser Ala 465

The open reading frame of this gene is shown in SEQ ID NO: 9. As shownin SEQ ID NO: 10, the entire portion is coded as a preprotein of 488amino acids, of which the N-terminal 22 residues are assumed to be thepre-region and the remaining 466 residues correspond to the matureprotein (cf. SEQ ID NO: 8).

The invention is not only particularly limited to a polypeptide havingan activity to act upon a disaccharide glycoside to release saccharidesfrom the disaccharide glycoside in disaccharide unit and a nucleotidewhich encodes the same, but also includes a more longer polypeptidecomprising the former polypeptide (e.g., precursor) and a nucleotidewhich encodes the same.

SEQ ID NO: 9 ggcgacacca gaaagcaacc aagagcacga cacggactta ttctctttg acaatg 56                                                           Met cgtata tct gtc ggt gct ctg ctt ggc ttg aca gcc ctg agt cat gcc 104 Arg IleSer Val Gly Ala Leu Leu Gly Leu Thr Ala Leu Ser His Ala    −20               −15                 −10 aca aca gag aaa cga gcc gcc tctgct tcg gct tac tgt tcc aac tcg 152 Thr Thr Glu Lys Arg Ala Ala Ser AlaSer Ala Tyr Cys Ser Asn Ser  −5              −1   1               5                  10 gcc ggc aac tac aag ctg tcc tcc atc gca gct ccg gttcaa ggg gcc 200 Ala Gly Asn Tyr Lys Leu Ser Ser Ile Ala Ala Pro Val GlnGly Ala              15                  20                  25 gga aacccc ggc tcg gaa tcg acc tgg caa ttg acc gtt gac gac act 248 Gly Asn ProGly Ser Glu Ser Thr Trp Gln Leu Thr Val Asp Asp Thr          30                 35                  40 tcg tcc ggt cac aaa cag acg atagtt ggg ttc ggt gct gct gtc act 296 Ser Ser Gly His Lys Gln Thr Ile ValGly Phe Gly Ala Ala Val Thr      45                  50                 55 gat gcc acg gtc acc tcg ttc aac act ttg tcc gcc tccgtg ctg caa 344 Asp Ala Thr Val Thr Ser Phe Asn Thr Leu Ser Ala Ser ValLeu Gln  60                  65                  70                  75gac ttg ctc aat aaa ctg atg aca cct gcc ggg gcg aac ttt gct ttg 392 AspLeu Leu Asn Lys Leu Met Thr Pro Ala Gly Ala Asn Phe Ala Leu                 80                  85                  90 atg cga catact att ggg gct tcg gat ctg tcc ggt gac cca gcc tac 440 Met Arg His ThrIle Gly Ala Ser Asp Leu Ser Gly Asp Pro Ala Tyr              95                100                 105 acg tac gat gac aat ggt ggg aaagcg gat ccg tca ctg tcg gga ttc 488 Thr Tyr Asp Asp Asn Gly Gly Lys AlaAsp Pro Ser Leu Ser Gly Phe         110                 115                120 aac ctg ggg gac cgc gga acg gct atg gcc aag atg ttggca aca atg 536 Asn Leu Gly Asp Arg Gly Thr Ala Met Ala Lys Met Leu AlaThr Met     125                 130                 135 aag tct ctg cagccc aac ctc aag atc ctc ggc tct ccc tgg agt gca 584 Lys Ser Leu Gln ProAsn Leu Lys Ile Leu Gly Ser Pro Trp Ser Ala 140                 145                150                 155 cca gga tgg atg aag ctg aac ggggtc ctt gat ggc aat acg aac aac 632 Pro Gly Trp Met Lys Leu Asn Gly ValLeu Asp Gly Asn Thr Asn Asn                 160                 165                170 aac aac ttg aac gat gga tac cta acc agt ggg ggg accggt agt acg 680 Asn Asn Leu Asn Asp Gly Tyr Leu Thr Ser Gly Gly Thr GlySer Thr             175                 180                 185 ggg tatgcc agt caa ttc gcg cag tac ttt gtc aag tac att cag gcc 728 Gly Tyr AlaSer Gln Phe Ala Gln Tyr Phe Val Lys Tyr Ile Gln Ala         190                195                 200 tat aag aat ctc ggt gct cac gtcgac gcg att acc atc cag aac gag 776 Tyr Lys Asn Leu Gly Ala His Val AspAla Ile Thr Ile Gln Asn Glu     205                 210                215 ccg ctg ttc agc tca gcg ggc tat ccc acc atg tat gtctac gat tat 824 Pro Leu Phe Ser Ser Ala Gly Tyr Pro Thr Met Tyr Val TyrAsp Tyr 220                 225                 230                 235gag tcg gca cag ctg atc cag aac tac atc ggc ccc gct ctt gcc agc 872 GluSer Ala Gln Leu Ile Gln Asn Tyr Ile Gly Pro Ala Leu Ala Ser                240                 245                 250 gcg ggg ctagat acg gaa atc tgg gct tat gac cac aac aca gat gtc 920 Ala Gly Leu AspThr Glu Ile Trp Ala Tyr Asp His Asn Thr Asp Val             255                260                 265 ccg tcg tac ccc cag act gtc cttaac cag gcc ggt cag tac gtc aag 968 Pro Ser Tyr Pro Gln Thr Val Leu AsnGln Ala Gly Gln Tyr Val Lys         270                 275                280 tcg gtg gcc tg cac tgc tac gct ccc aac gtc gac tggacc gtg ctc 1016 Ser Val Ala Trp His Cys Tyr Ala Pro Asn Val Asp Trp ThrVal Leu     285                 290                 295 agc cag ttc cacaac aca aac cct gga gtgaag caa tat atg acc gag 1064 Ser Gln Phe His AsnThr Asn Pro Gly Val Lys Gln Tyr Met Thr Glu 300                 305                310                 315 tgc tgg act cca gca tct ggc gcatgg cat cag gcg gcg gac ttc acc 1112 Cys Trp Thr Pro Ala Ser Gly Ala TrpHis Gln Ala Ala Asp Phe Thr                 320                 325                330 atg ggt ccc ctg cag aac tgg gcc tcg gga gtg gca gcatgg act ctg 1160 Met Gly Pro Leu Gln Asn Trp Ala Ser Gly Val Ala Ala TrpThr Leu             335                 340                 345 gga accaac gct cag gat ggt ccg cat ctg tcc act ggc ggc tgc gcg 1208 Gly Thr AsnAla Gln Asp Gly Pro His Leu Ser Thr Gly Gly Cys Ala         350                355                 360 aca tgt caa ggc ttg gtg acc atcaac aac gga gga tac acg ctc aac 1256 Thr Cys Gln Gly Leu Val Thr Ile AsnAsn Gly Gly Tyr Thr Leu Asn     365                 370                375 acc gca tac tac atg atg gcg caa ttc agc aag ttc atgccg cct ggt 1304 Thr Ala Tyr Tyr Met Met Ala Gln Phe Ser Lys Phe Met ProPro Gly 380                 385                 390                 395gcg att gtg ctc aat ggc agt ggc agc tac acg tac tct ggc gga ggc 1352 AlaIle Val Leu Asn Gly Ser Gly Ser Tyr Thr Tyr Ser Gly Gly Gly                400                 405                 410 ggt atc cagtcc gtg gct tcc ttg aat ccc gat gga acc cgc act gtg 1400 Gly Ile Gln SerVal Ala Ser Leu Asn Pro Asp Gly Thr Arg Thr Val             415                420                 425 gtt att gaa aac act ttt ggc aatgat gtc tat gtg act gtc act atg 1448 Val Ile Glu Asn Thr Phe Gly Asn AspVal Tyr Val Thr Val Thr Met         430                 435                440 aag agc ggg cag aag tgg agt ggg aac gcc cct agc caatcc gtg act 1496 Lys Ser Gly Gln Lys Trp Ser Gly Asn Ala Pro Ser Gln SerVal Thr     445                 450                 455 acc tgg gtt cttcca tct gct tga aaagagtgta gtttcagatg gttagatatg 1550 Thr Trp Val LeuPro Ser Ala 460                 465 tattgaagag tagcgcttgg agacatcaatagcctttttc taattacatg tcgtgcagct 1610 tccaaaaaaa aaaaaaaaaa aaaaaaaaaaaactcga 1647 SEQ ID NO: 10 Met Arg Ile Ser Val Gly Ala Leu Leu Gly LeuThr Ala Leu Ser His   1               5                  10                 15 Ala Thr Thr Glu Lys Arg Ala Ala Ser Ala Ser Ala TyrCys Ser Asn              20                  25                  30 SerAla Gly Asn Tyr Lys Leu Ser Ser Ile Ala Ala Pro Val Gln Gly          35                 40                  45 Ala Gly Asn Pro Gly Ser Glu SerThr Trp Gln Leu Thr Val Asp Asp      50                  55                 60 Thr Ser Ser Gly His Lys Gln Thr Ile Val Gly Phe GlyAla Ala Val 65                  70                  75                 80 Thr Asp Ala Thr Val Thr Ser Phe Asn Thr Leu Ser AlaSer Val Leu                  85                  90                  95Gln Asp Leu Leu Asn Lys Leu Met Thr Pro Ala Gly Ala Asn Phe Ala            100                 105                 110 Leu Met Arg HisThr Ile Gly Ala Ser Asp Leu Ser Gly Asp Pro Ala         115                120                 125 Tyr Thr Tyr Asp Asp Asn Gly GlyLys Ala Asp Pro Ser Leu Ser Gly     130                 135                140 Phe Asn Leu Gly Asp Arg Gly Thr Ala Met Ala Lys MetLeu Ala Thr 145                 150                 155                160 Met Lys Ser Leu Gln Pro Asn Leu Lys Ile Leu Gly SerPro Trp Ser                 165                 170                 175Ala Pro Gly Trp Met Lys Leu Asn Gly Val Leu Asp Gly Asn Thr Asn            180                 185                 190 Asn Asn Asn LeuAsn Asp Gly Tyr Leu Thr Ser Gly Gly Thr Gly Ser         195                200                 205 Thr Gly Tyr Ala Ser Gln Phe AlaGln Tyr Phe Val Lys Tyr Ile Gln     210                 215                220 Ala Tyr Lys Asn Leu Gly Ala His Val Asp Ala Ile ThrIle Gln Asn 225                 230                 235                240 Glu Pro Leu Phe Ser Ser Ala Gly Tyr Pro Thr Met TyrVal Tyr Asp                 245                 250                 255Tyr Glu Ser Ala Gln Leu Ile Gln Asn Tyr Ile Gly Pro Ala Leu Ala            260                 265                 270 Ser Ala Gly LeuAsp Thr Glu Ile Trp Ala Tyr Asp His Asn Thr Asp         275                280                 285 Val Pro Ser Tyr Pro Gln Thr ValLeu Asn Gln Ala Gly Gln Tyr Val     290                 295                300 Lys Ser Val Ala Trp His Cys Tyr Ala Pro Asn Val AspTrp Thr Val 305                 310                 315                320 Leu Ser Gln Phe His Asn Thr Asn Pro Gly Val Lys GlnTyr Met Thr                 325                 330                 335Glu Cys Trp Thr Pro Ala Ser Gly Ala Trp His Gln Ala Ala Asp Phe            340                 345                 350 Thr Met Gly ProLeu Gln Asn Trp Ala Ser Gly Val Ala Ala Trp Thr         355                360                 365 Leu Gly Thr Asn Ala Gln Asp GlyPro His Leu Ser Thr Gly Gly Cys     370                 375                380 Ala Thr Cys Gln Gly Leu Val Thr Ile Asn Asn Gly GlyTyr Thr Leu 385                 390                 395                400 Asn Thr Ala Tyr Tyr Met Met Ala Gln Phe Ser Lys PheMet Pro Pro                 405                 410                 415Gly Ala Ile Val Leu Asn Gly Ser Gly Ser Tyr Thr Tyr Ser Gly Gly            420                 425                 430 Gly Gly Ile GlnSer Val Ala Ser Leu Asn Pro Asp Gly Thr Arg Thr         435                440                 445 Val Val Ile Glu Asn Thr Phe GlyAsn Asp Val Tyr Val Thr Val Thr     450                 455                460 Met Lys Ser Gly Gln Lys Trp Ser Gly Asn Ala Pro SerGln Ser Val 465                 470                 475                480 Thr Thr Trp Val Leu Pro Ser Ala                 485

EXAMPLE 9

Confirmation of the Expression of β-Primeverosidase in Mold

a) Construction of Expression Cassette

In order to verify whether or not the cloned gene is the primeverosidasegene, expression of the thus obtained DNA was confirmed. Using anAspergillus oryzae Taka-amylase gene-containing plasmid pTG-Taa (Kato M,Aoyama A, Naruse F, Kobayashi T and Tsukagoshi N (1997), An Aspergillusnidulans nuclear protein, An CP, involved in enhancement of Taka-amylaseA gene expression binds to the CCAAT-containing taaG2, amdS and gatApromoters., Mol. Gen. Genet., 254: 119–126) as the template, a fragmentwas obtained by amplifying it by PCR using a primer TAA5′

SEQ ID NO: 11

sense primer:

5′-GGGCCTGCAGGAATTCATGGTGTT-3′

and a primer TP3′

SEQ ID NO: 12

antisense primer:

5′-CGAGCCGGGGTTTCCGTCCGCAGGCGTTGC-3′.

<PCR reaction solution> template DNA solution (50 μg/ml) 1 μl 50 μMsense primer 1 μl 50 μM antisense primer 1 μl sterilized water 22 μlPremix Taq (EX Taq Version TaKaRa) 23 μl <PCR reaction conditions> Stage1: denaturation (95° C., 1 minute) 1 cycle Stage 2: denaturation (95°C., 1 minute) 30 cycles annealing (55° C., 1 minute) elongation (72° C.,1 minute) Stage 3: elongation (72° C., 5 minutes) 1 cycle

Also, a fragment was obtained by amplifying it by PCR using theDNA-containing plasmid pAFPri as the template and using a primer dPC5′

SEQ ID NO: 13

sense primer:

5′-GCAACGCCTGCGGACGGAAACCCCGGCTCG-3′

and a primer PC3′

SEQ ID NO: 14

antisense primer:

5′-GCGCAAGCTTGGAAGCTGCACGACATGTAA-3′.

In addition, after recovering and mixing respective fragments, afragment was obtained by amplifying it by PCR using the primer TAA5′ andprimer PC3′. This fragment contains a sequence corresponding to a regionof from the Aspergillus oryzae Taka-amylase promoter to the N-terminal5th amino acid of the mature protein and a sequence corresponding to aregion of from the N-terminal 28th amino acid (glycine) to theC-terminal of the mature β-primeverosidase protein. An Sse8387I site hasbeen introduced into the upstream of the thus obtained fragment, and aHindIII site into its downstream. The fragment was recovered by treatingwith restriction enzymes Sse8387I and HindIII.

Regarding the terminator region, a fragment was obtained by amplifyingit by PCR using pTG-Taa as the template and using a primer TAAH

SEQ ID NO: 15

sense primer:

5′-GCGCAAGCTTTGAAGGGTGGAGAGT-3′

and a primer TAA3′

SEQ ID NO: 16

antisense primer:

5′-GCGCCCTGCAGGTCTAGAATTCCTAGTGGTT-3′.

A HindIII site has been introduced into the upstream of the thusobtained fragment, and an Sse8387I site into its downstream. Thefragment was recovered by treating with restriction enzymes HindIII andSse8387I.

A plasmid pTG1 containing orotidine-5′-phosphate decarboxylase gene(pyr4) as a marker gene (Kato M (1997), Mol. Gen. Genet., 254: 119-126)was treated with the restriction enzyme Sse8387I and with alkalinephosphatase and then recovered.

Plasmids pAFPriE1 (forward direction to the direction of the markergene) and pAFPriE2 (reverse direction) were obtained by connecting these3 fragments.

b) Acquisition of Transformant

An orotidine-5′-phosphate decarboxylase (PyrG) producing strainAspergillus nidulans G191 (Kato M (1997), Mol. Gen. Genet., 254:119-126) was inoculated into a complete medium (2% malt extract, 0.1%peptone, 2% glucose, 0.1% uridine, 2 μg/ml p-aminobenzoic acid, pH 6.5)and cultured at 30° C. for 18 hours on a shaker. The cells werecollected by filtration, suspended in a protoplast solution (0.8 M NaCl,10 mM NaH₂PO₄, 20 mM CaCl₂, 3.75 mg/ml Novozyme 234) and then treated at30° C. for 1 hour on a shaker. The resulting protoplasts were recoveredby filtration and centrifuged at 1,500 rpm for 5 minutes to obtain theprotoplasts as the precipitate. This precipitate was suspended in 0.8 MNaCl solution and centrifuged at 1,500 rpm for 5 minutes to collect theprecipitate. This was again suspended in 0.8 M NaCl/50 mM CaCl₂ solutionand centrifuged at 1,500 rpm for 5 minutes to collect the precipitate. Aprotoplast solution was obtained by suspending this in an appropriateamount of 0.8 M NaCl/50 mM CaCl₂ solution. Next, 50 μl of thisprotoplast solution was mixed with 20 μg of a DNA solution and 12.5 μlof a PEG solution (25% PEG 6000/50 mM CaCl₂/10 mM Tris-HCl (pH 7.5)) andthen allowed to stand for 20 minutes on ice. Next, 0.5 ml of PEGsolution was added and then the mixture was allowed to stand for 5minutes on ice. Next, 1 ml of 0.8 M NaCl/50 mM CaCl₂ solution was addedand mixed. A 0.5 ml portion of this mixed solution was mixed with 15 mlof 2% agar-containing regeneration medium (0.6% NaNO₃, 11 mM KH₂PO₄, 7mM KCl, 1.2 M sorbitol, 0.05% MgSO₄.7H₂₀, 1% glucose, 2 μg/mlp-aminobenzoic acid, pH 6.5) which had been incubated at 50° C. inadvance, solidified in Petri dishes and then cultured at 37° C. for 3days.

This was carried out on the plasmid DNA of each of pTG1, pAFPriE1 andpAFPriE2.

Colonies formed on the plates were isolated by single spore separation.A total of 15 transformant strains were obtained from pTG1, and 23strains from pAFPriE1 and 13 strains from pAFPriE2.

c) Evaluation of Transformants

Evaluation of transformants was carried out on 8 strains obtained frompTG1, 18 strains from pAFPriE1 and 12 strains from pAFPriE2. Eachtransformant was inoculated into an enzyme production confirming medium(1% polypeptone, 0.5% KH₂PO₄, 0.1% NaNO₃, 0.05% MgSO₄.7H₂O, 2% maltose,4 μg/ml p-aminobenzoic acid, 0.1% trace element solution) (Core DJ.,Biochem., Biophys. Acta, 1996, vol., 113, pp. 51–56) and cultured at 30°C. for 96 hours on a shaker. The culture broths were sampled after 48,72, and 96 hours and filtered, and the resulting filtrates were checkedfor the activity. The enzyme activity was not found in the transformantsobtained from pTG1 and pAFPriE2 but the enzyme activity was confirmed in5 transformant strains obtained from pAFPriE1.

EXAMPLE 10

Comparison with Plant Gene by a Hybridization Method

Using the gene of an enzyme similar to the tea-derived diglycosidase asthe probe, an examination was carried out to know if a gene having asimilar structure is present on the chromosome of the microorganisms inwhich the presence of diglycosidase had been confirmed by us.Preparation of gene fragment of an enzyme similar to the tea-deriveddiglycosidase was carried out with reference to the report by Sakata,Mizutani et al. (The 73rd Annual Meeting of Agricultural ChemicalSociety of Japan) and Japanese Patent Application No. Hei. 11-56299.

Preparation of microorganism-derived chromosome was carried out in thefollowing manner.

Preparation of chromosomes from yeast and fungi was carried out inaccordance with the method described in Molecular and Cellular Biology,Vol. 3, pp. 1430–1439 (1983). Preparation of chromosomal DNA frombacteria was carried out in accordance with the method of Saito andMitsuura (Biochim. Biophys. Acta, Vol. 72, pp. 619–629, 1963).Preparation of chromosomal DNA from actinomycetes was carried out inaccordance with the method of Iefuji et al. (Biosci. Biotec. Biochem.,Vol. 60, pp. 1331–1338, 1996).

A 10 μg portion of each of the thus obtained various chromosomal DNApreparations was digested with BamHI in the case of Aspergillusfumigatus, Aspergillus oryzae, Aspergillus niger, Aspergillus aculaetus,Penicillium lilacinum, Penicillium decumbence, Penicillium multicolor,Talaromyces emersonii, Mortierella vinacea, Cryptococcus albidus,Corynebacterium ammoniagenes, Corynebacterium glutamicum, Microbacteriumarborescens and Penicillium rugolosum, or with EcoRI in the case ofRhizopus oryzae, Rhizomucor pusillus, Rhizomucor miehei and Actinoplanesmissouriensis, and the resulting digest was applied to a 1% agarose gelelectrophoresis. As a control, the gene fragment of an enzyme similar tothe tea-derived diglycosidase used as the probe was also subjected tothe same gel electrophoresis. After the electrophoresis, DNA sampleswere blotted on a nylon membrane and hybridization was carried out usinga labeled gene fragment p of an enzyme similar to the tea-deriveddiglycosidase (structural gene moiety of matured plant primeverosidasegene) as the probe, using DIG System Kit (Boehringer Mannheim) inaccordance with the instruction attached thereto. As a result, when thedetection was carried out under hybridization conditions (5×SSC, 1%blocking agent, 0.1% N-lauroylsarcosine sodium, 0.02% SDS, 68° C.,overnight) and washing conditions (6×SSC, 0.1% SDS, room temperature, 5min.×2 and 6×SSC, 0.1% SDS, 45° C., 15 min.×2), a signal was obtained ata position where the plant gene was blotted, but the signal was notobserved at any other position where the microorganism-derived genomewas blotted. Thus, it is considered that the microorganism-deriveddiglycosidase gene has a structure which is different from the plantprimeverosidase gene.

On the other hand, using the Aspergillus fumigatus IAM 2020diglycosidase gene of the invention obtained in Example 8 as the probe,an examination was carried out by the same methods and conditions toknow if a gene having a similar structure is present on the chromosomeof the microorganisms in which the presence of diglycosidase had beenconfirmed by us. As a result, the signal was detected in thesemicroorganisms.

In addition, it was able to detect the signal in Aspergillus oryzae,Aspergillus niger, Aspergillus aculeatus, Penicillium multicolor,Penicillium lilacinum, Corynebacterium ammoniagenes and Corynebacteriumglutamicum, even under more stringent washing conditions (5×SSC, roomtemperature, 10 min. and 4×SSC, 68° C., 30 min.).

EXAMPLE 11

Activity of the Diglycosidase to Hydrolyze Isoflavone in IsoflavoneGlycosides

As shown in the following table, glucosides and modified glucosides ofacetylglucosides and malonylglucosides, are present in isoflavoneglycosides. The activity of diglycosidase to hydrolyze the acetyl typeand malonyl type glucosides, namely the aglycon releasing activity, wasexamined.

Con- Isoflavone M. wt. R₁ R₂ R₃ centration Glycitin 446.4 H OCH₃ H 2 mMGenistin 432.4 OH H H 2 mM Daidzin 416.4 H H H 2 mM Acetylglycitin 458.4H OCH₃ COCH₃ 2 mM Acetylgenistin 474.7 OH H COCH₃ 2 mM Acetyldaidzin458.4 H H COCH₃ 2 mM Malonylglycitin 502.4 H OCH₃ COCH₂COOH 2 mMMalonylgenistin 518.4 OH H COCH₂COOH 2 mM Malonyldaidzin 502.4 H HCOCH₂COOH 2 mM R₁ to R₃ correspond to the following structural formula.

Each of acetylglycitin, acetylgenistin, acetyldaidzin, malonylglycitin,malonylgenistin and malonyldaidzin (produced by Fujicco, available fromNakalai Tesque) was allowed to react with a diglycosidase enzymesolution prepared from Asp. fumigatus or Pen. multicolor or with analmond-derived glucosidase (mfd. by Sigma) under the followingconditions.

Each of the enzymes was diluted with 20 mM acetate buffer (pH 4.0) toadjust its activity to 1.88 AU/ml, and then 2 mM of each isoflavone(12.5 μg), 20 mM of acetate buffer (87.5 μl) and each enzyme solution(25 μl) were mixed to carry out the reaction at 55° C. After 1, 3 and 6hours of the reaction, samples were taken out in 25 μl portions, andeach of the samples was mixed with 75 μl of methanol and 900 μl ofwater, filtered through a filter (0.2 μm) and then further diluted 2.5times with water. A 1 ml portion thereof was analyzed by HPLC (HPLCconditions; column: ODS 80TM (Tosoh), eluent: a mixed solution ofacetonitrile and 10% acetic acid, separation under linear densitygradient).

As a result, it was revealed that the glucosidase (Sigma) hardlyhydrolyzed the modified glucoside substrates, but both diglycosidaseenzymes hydrolyzed all of the modified glucosides efficiently andreleased the aglycon.

EXAMPLE 12

Preparation of an Aroma Component Precursor, Eugenyl Primeveroside

A 2 kg portion of fresh leaves of Camellia sasanqua were extracted withhot water at 100° C. for 10 minutes, and the extract was applied to acolumn packed with Diaion HP20 (mfd. by Mitsubishi Chemical) to adsorbeugenyl primeveroside thereon. The column was washed with about 2 timesthe bed volume of deionized water and 20% methanol and then the adsorbedeugenyl primeveroside was eluted with 100% methanol. Thereafter, thethus recovered methanol solution containing eugenyl primeveroside wasconcentrated to crystallize eugenyl primeveroside which was thenrecovered using a glass filter.

A 1 ml portion of the crude enzyme concentrate obtained in Example 4 wasmixed with 1 ml of the eugenyl primeveroside solution adjusted to 5mg/ml with 20 mM phosphate buffer (pH 6.0), the mixture was incubated at37° C. for 24 hours and then the formation of aroma was examined by asensory test (10 panel). As a result, formation of the eugenol-specificaroma was confirmed in all of the cases in which the crude enzymeconcentrates of two Aspergillus niger strains and Aspergillus fumigatuswere used.

In a case in which the crude enzyme concentrates were used after theirheat-treatment (100° C., 10 minutes), the eugenol-specific aroma was notobserved. Accordingly, it was found that these crude enzyme extractshave a function to release the aroma component aglycon from glycosidessuch as eugenyl primeveroside.

EXAMPLE 13

Release of Disaccharide from pNP-primeveroside by Purified Enzyme

A 0.3 AU portion of the purified enzyme solution obtained in Example 6and the aforementioned pNP-primeveroside were incubated at 37° C. for 24hours to examine release of disaccharide by TLC. As a result, a spot wasobserved at the same position of primeverose so that release of adisaccharide was confirmed. Such a spot was not found by theheat-treated purified enzyme solution used as a control. Thus, it wasrevealed that the purified enzyme has a function to release adisaccharide from a disaccharide glycoside.

EXAMPLE 14

Release of Disaccharide and Formation of Aroma from EugenylPrimeveroside by Purified Enzyme

A 0.3 AU portion of the purified enzyme solution obtained in Example 6and the aforementioned pNP-primeveroside were incubated at 37° C. for 24hours to examine release of disaccharide by TLC. As a result, a spot wasobserved at the same position of primeverose so that release of adisaccharide was confirmed. In addition, when the reaction solution wasanalyzed by a gas chromatography, release-of eugenol as the aglycon ofeugenyl primeveroside glycoside was confirmed, and the release ofeugenol was also confirmed by a sensory test. These were not found inheat-inactivated enzyme solution. Thus, it was revealed that thepurified enzyme forms aroma by acting upon an aroma precursor such aseugenyl primeveroside.

EXAMPLE 15

Release of Disaccharide from Pigment Glycoside

Release of disaccharide was examined using a disaccharide glycoside,ruberythric acid, as the substrate. Ruberythric acid was prepared byadsorbing a water extract of Rubia tinctorum L. root powder for staininguse (available from Tanaka Senshoku Ten) to HP-20 column, washing thecolumn with 50% methanol, and then eluting the compound with 100%methanol and evaporating the eluate to dryness using an evaporator. Asubstrate prepared by dissolving the thus recovered ruberythric acid ina phosphate buffer to a concentration of 5 mg/ml was mixed with thecrude enzyme solution (0.3 AU) shown in Example 4 or the purified enzymesolution shown in Example 6 and incubated at 37° C. for 24 hours, andthen the reaction solution was analyzed by TLC. As a result, release ofthe disaccharide primeverose and the aglycon alizarin was observed bythe crude enzyme solution and purified enzyme solution.

Using the diglycosidase preparations derived from various microorganismsshown in Example 5, their ability to hydrolyze various disaccharideglycosides was examined using TLC. As a result, it was revealed that thediglycosidase acts upon not only the primeveroside glycosides but alsovarious other disaccharide glycosides analogous to the primeverosideglycosides, including rutinose glycosides such as naringin and rutin,gentiobiose glycosides, arabinofuranosyl glycosides and apiofuranosylglycosides, and thereby releases disaccharides and produces respectivefree aglycons.

EXAMPLE 17

Improvement of Tea Extract Aroma

Using the Aspergillus fumigatus enzyme solutions shown in Examples 4 and6, their function to increase aroma components of green tea, black teaand oolong tea was examined. Each tea extract was mixed with 1.88 AU ofthe enzyme and incubated at 55° C. for 24 hours and then increase in thearoma was examined by a gas chromatography under the aforementionedconditions. As a result, it was found that aroma components such as1-hexanol, 3-hexen-1-ol, benzaldehyde, linalool, methyl salicyanate,geraniol and benzyl alcohol were increased. Increase in the aroma wasalso found by a sensory test.

EXAMPLE 18

Improvement of Fruit Juice Aroma

Using the Aspergillus fumigatus and Penicillium multicolor enzymesolutions shown in Examples 4 and 5, 1.88 AU of each of the enzyme wasadded to a fruit juice such as of grape, orange, apple, prune or nectarand incubated at 37° C. for 24 hours and then the aroma was analyzed bya gas chromatography. As a result, increase in the aroma components suchas linalool was observed. Improvement of the aroma was also found in theenzyme-treated fruit juices by a sensory test.

EXAMPLE 19

Improvement of Wine Aroma

Using the various crude enzyme solutions shown in Example 4, 0.5 AU ofeach enzyme was added to red wine and white wine and incubated at 37° C.for 24 hours to examine improvement of the aroma by a sensory test. As aresult, improvement of the aroma was found in both cases.

EXAMPLE 20

When 1 ml of each of the various crude enzyme concentrates obtained inExamples 4 and 5 was mixed with 1 ml of a grape juice (commercialproduct: 100% fruit juice, concentrated and reduced) and then incubatedat 37° C. overnight (14 hours) to examine the aroma, the aroma wasclearly improved in comparison with a sample in which an acetate bufferwas added instead of the enzyme preparation. In addition, this functionwas not found when the crude enzyme concentrate was heat-treated at 100°C. for 10 minutes.

EXAMPLE 21

A 1 ml portion of each of the crude enzyme concentrates obtained inExample 4 was mixed with 1 ml of a commercially available orange juice(reduced concentrate) and incubated at 37° C. for 24 hours to examineformation of the aroma by a sensory test. As a result, improving effectof the aroma of the orange juice was found in the crude enzymeconcentrates derived from the two Aspergillus niger strains andAspergillus fumigatus. Such a function was not found when the crudeenzyme concentrate was heat-treated (100° C., 10 minutes).

EXAMPLE 22

An examination was carried out to find whether a sugar transfer indisaccharide unit is generated by diglycosidase.

The purified enzyme of Example 6 was diluted with deionized water toadjust the activity to E1.0 AU/ml, and acetonitrile (200 ml) containing2.5% phenethyl alcohol, 20 mM acetate buffer (250 μl) containing 10%primeverose and the enzyme solution (50 μl) were mixed to carry out thereaction at 55° C. The reaction was completed after 6 hours and thereaction solution was mixed with 500 μl of diethyl ether, stirred andthen centrifuged to remove free aglycon which was transferred into theether layer. By applying the water layer to a Diaion HP-20 column andpassing purified water through the column, free primeverose was removed.The disaccharide glycoside adsorbed to the resin was eluted withmethanol and concentrated to dryness. This was dissolved in 100 μl ofdeionized water, and a 20 μl portion thereof was spotted on a TLC plateto detect the reaction product (the developing solvent was ethylacetate:acetic acid:deionized water=3:1:1, acetic acid:methanol=1:4solution was sprayed thereto after the development and allowed to standat 105° C. for 10 minutes).

As a result, it was revealed that β-primeverosidase transfers thediglycoside to phenethyl alcohol and thereby forms a disaccharideglycoside.

INDUSTRIAL APPLICABILITY

An enzyme having a function to cut β-primeveroside and/or analogousdisaccharide glycoside in disaccharide unit or to hydrolyze modifiedglucosides can be provided by the invention as a novel enzyme usingmicroorganisms as its supply source, and it can be broadly used invarious types of food, medicaments, quasi drugs and the like by usingthe enzyme composition of the invention. For example, the aroma, pigmentand physiologically active component of food can be increased ordecreased.

1. An isolated polypeptide comprising the amino acid sequence of SEQ IDNO:8.
 2. An isolated polypeptide consisting of the amino acid sequenceof SEQ ID NO:8.